We have utilized hypomorphic suppression of the ATR checkpoint kinase to promote replication fork collapse into DSBs selectively in oncogene-expressing cells and those lacking the p53 tumor suppressor gene. Understanding the process of replication fork collapse is helpful in determining the ideal genetic contexts in which such therapies should be applied. Towards this objective, we have recently demonstrated that the AURKA-PLK1 pathway and the SUMO-targeted E3 ubiquitin ligase RNF4 are required for fork collapse and DSB formation when ATR is inhibited. In this grant proposal, we will 1) dissect the regulatory interrelationships between AURKA-PLK1, RNF4 and ATR in replication fork stability, 2) characterize the ubiquitination events mediated by the AURKA-PLK1 pathway and RNF4 that drive the disengagement of replication factors from the fork, and 3) determine if the amplification of AURKA activity and replisome sumoylation in human breast cancers promotes sensitivity to ATR/CHK1 inhibitors in cultured cells and orthotopically transplanted tumors in mice. These studies will advance our understanding of the causes of replication fork collapse upon ATR/CHK1 inhibition and investigate the prognostic value of amplified AURKA-PLK1 activity and replisome sumoylation in responsiveness to ATR/CHK1 inhibitor treatments.